(B) Multiple sequence alignment of EspFu from strains EPEC BA320, EHEC EDL933 (GenBank accession no. Martins et al. This content is usually distributed under the terms Polyphyllin B of the Creative Commons Attribution 4.0 International license. FIG?S2. Combination of EspFu and TirS is usually associated with increased bacterial attachment and pedestal formation. (A) Immunofluorescence assay showing production and translocation of Tir to the host cell. HeLa cells were infected with the indicated strains, fixed, and then labeled with rabbit anti-Tir polyclonal serum (red), phalloidin-FITC (actin, green), and DAPI (bacteria and cell nuclei, blue). Scale bar, 20 m. (B) Quantification of FAS showing the percentage of cells with EPEC forming actin pedestals. The number of cells with pedestals was enumerated in multiple fields, with each field containing at least 20 cells. (C) Quantification of bacterial adherence showing the number of recovered bacteria (CFU/well) after plating cell lysates onto LB agar plates supplemented with appropriate antibiotics. Error bars represent means the SD from three biological replicates. Statistical significance was determined by using an unpaired Student test. *, < 0.05; **, < 0.01. Download FIG?S2, TIF file, 2.5 MB. Copyright ? 2020 Martins et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S3. Activation of proinflammatory genes by EPEC depends on the mechanism of pedestal formation rather than an enhanced bacterial association with the epithelium. (A and E) FAS assay on HeLa cells infected with BA320 (WT, MOI of 10), KOct1 (MOI of 10), KOct2 (MOI of 100), and KO (MOI of 100) strains. Scale bar, 20 m. (B and F) Quantification of FAS showing the percentage of cells with EPEC-forming actin pedestals. (C and G) Quantification of bacterial adherence showing the Polyphyllin B number of recovered bacteria (CFU/well) after plating cell lysates onto LB agar plates supplemented with appropriate antibiotics. Error bars represent means the SD from six biological replicates. (D and H) qRT-PCR analysis of the expression levels of CXCL1 and IL8 genes in HeLa cells infected with similar bacterial loads of EPEC strains. Data were normalized to B2M (endogenous control) and presented as means the SD from three biological replicates. Statistical significance was determined by using an unpaired Student test. *, < 0.05; **, < 0.01; ***, < 0.0001; ns, not significant. Download FIG?S3, TIF file, 2.9 MB. Copyright ? 2020 Martins et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 Rabbit polyclonal to LACE1 International license. FIG?S4. IPA analysis of TNF receptor 2 (TNFR2; A) and interleukin-6 (IL-6; B) signaling pathways activated in cells infected with pedestal-forming EPEC strains. Genes that showed differential expression are highlighted in color. Color intensity reflects magnitude of change (red, upregulated; green, downregulated). Genes without color were not affected by the treatment. Solid lines represent direct interactions. Download FIG?S4, TIF file, 1.2 MB. Copyright ? 2020 Martins et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. FIG?S5. Molecular networks of hypoxia-induced factor 1 (HIF1A; A), interleukin-1 (IL1B; B), and tumor necrosis factor (TNF; B) identified by IPA in cells infected with pedestal-forming EPEC strains. Genes that showed differential expression are highlighted in color. Color intensity reflects magnitude of change (red, upregulated; green, downregulated). Genes without color were not affected by the treatment. Solid lines represent direct interactions and dashed lines indirect interactions. Download FIG?S5, TIF file, 2.1 MB. Copyright ? 2020 Martins et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license. TABLE?S1. Bacterial strains, plasmids, and oligonucleotide primers used in this study. Download Table?S1, DOCX file, 0.03 MB. Copyright ? 2020 Martins et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 Polyphyllin B International license. TABLE?S2. List of differentially expressed genes (DEGs) in each comparison. Download Table?S2, DOCX file, 0.05 MB. Copyright ? 2020 Martins et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 Polyphyllin B International license. VIDEO?S1. Live-cell imaging of HeLa cells.